Nucleic acid amplification has been widely used for research and molecular diagnostics. PCR (polymerase chain reaction) is a traditional method for nucleic acid amplification which requires thermocycling. Heat denaturation during PCR thermocycling is a powerful and important step to uniformly separate DNA duplexes, generating single-stranded templates for subsequent primer annealing. Several platforms exist for isothermal amplification, including SDA (strand displacement amplification), TMA (transcription-mediated amplification), RCA (rolling cycle amplification), LAMP (loop-mediated amplification), NASBA (nucleic acid sequence-based amplification), and HDA (helicase-dependent amplification). HDA is different from other isothermal technologies because the HDA technology employs a helicase to separate the double-stranded nucleic acid. It is desirable to improve the speed and sensitivity of nucleic acid amplification technologies with high speed and high sensitivity, particularly for use in research and diagnostic applications.
Several methods have been developed to improve speed and sensitivity for PCR. Some of the methods that can improve PCR performance have also been successful for improving HDA performance. However, HDA is different from PCR, as HDA can be performed isothermally and relies on a helicase to separate the strands of double-stranded nucleic acids. Several specific methods have been applied to improve HDA. For example, previous studies have found that the protein concentrations of DNA polymerase and helicase can be optimized to improve the performance of HDA. Although increasing amounts of enzymes (helicases and DNA polymerases) can increase detection speed, the Limit-of-Detection (LoD), the minimum number of target nucleic acids required for reliable detection, generally remains unchanged at, for example, 5-50 copies.
HDA uses helicase(s) to separate a DNA duplex. Helicase is not a sequence-specific protein and therefore does not specifically recognize a target region. The efficiency of the helicase separating the double-stranded nucleic acid, especially in the target region specified by forward and reverse primers, is therefore a rate-limiting factor. Based on the unwinding direction, helicases can be grouped into two major types: the 3′-5′ helicases and the 5′-3′ helicases. The well-studied E. coli UvrD helicase unwinds DNA in a 3′ to 5′ direction (Matson, S. W. J. Biol. Chem., 261, 10169-10175. (1986)). Some helicases can unwind duplex nucleic acid from blunt ends; advantages of using such helicases are described in U.S. Pat. No. 7,282,328. Other helicases require a single-stranded “tail” on the substrate duplex, generally a 3′ tail for 3′-5′ helicases and a 5′ tail for the 5′-3′ enzymes.